JP2018017657A - Method for analyzing cell secretion and multi-well plate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for analyzing a secretion of a living cell with a high throughput.SOLUTION: The present invention relates to a method for analyzing a chemical substance 3 secreted from a cell 1. According to the method, a chemical substance secreted from a cell contained in a confinement region 11 of a well 10 is analyzed by chemical analysis means in a measurement region 12 leading to the confinement region, the measurement region 12 blocking entrance of cells into the region 12.SELECTED DRAWING: Figure 1

Description

  The present disclosure relates to an analysis method for analyzing secretions from cells, and a multi-well plate used for analysis of cell secretions, and in particular, to analyze secretions secreted from cells at high throughput. The present invention relates to a method for analyzing cell secretions that can be performed and a multiwell plate that can be used for the analysis.

  Secretions from cells constitute blood, urine, puncture fluid, etc., and quantitative analysis of chemical substances such as enzymes, lipids, and proteins contained in these secretions enables diagnosis, treatment, and progress of disease states. Observations are carried out. Conventionally, these chemical substances have been analyzed as an aggregate of secretions from various types of cells, but in order to improve detection accuracy, quantitative analysis of secretions at the level of one cell is possible. Is required.

  As a method of quantitatively analyzing secretions from a single cell, a multi-well plate with a plurality of wells having a size and shape capable of accommodating only one cell was prepared, and the cell was suspended. A method has been proposed in which an aqueous solution is supplied onto the multi-well plate to create a state in which one cell is confined in one well, and the liquid in the well in which the cell is confined is collected and analyzed. (See Patent Document 1 and Non-Patent Document 1).

JP 2014-233208 A

H. Fujita, T. Esaki, T. Masujima, A. Hotta, SH Kim, H. Noji, and TM Watanabe, "Comprehensive chemical secretory measurement of single cells trapped in a micro-droplet array with mass spectrometry," RSC Adv. 5, 16968-16971 (2015).

  According to the conventional analysis method described above, a space containing only one cell is created, and the secretions secreted into the space are individually collected, so that only the secretions from one cell are acquired and mass spectrometry is performed. The component can be accurately analyzed by a method or the like.

  However, in the conventional analysis method described above, the aqueous solution in the well containing the cells is collected using a syringe pump with a metal-coated glass microneedle, which corresponds to a so-called destructive test, and the secretion from one cell. Once recovered, the secretion from the same cell cannot be recovered and analyzed again. In addition, the location of the glass microneedles must be determined while enlarging the micron-order well with a microscope, because it is necessary to collect the aqueous solution where the cells do not exist while confirming the position of the cells in the well. There is a limit to improving work efficiency. Furthermore, when performing mass spectrometry by ESI analysis, for example, it is necessary to atomize the collected secretion and introduce it into a measuring instrument, which requires additional preparation steps for analyzing the secretion.

  Therefore, the present application solves the above-described conventional problems, and a cell secretion analysis method that can analyze a secretion secreted by a cell in a state where the cell is alive and at a high throughput, and this analysis method An object of the present invention is to obtain a multiwell plate that can be suitably used for the above.

  In order to solve the above problems, the method for analyzing a cell secretion disclosed in the present application is an analysis method for analyzing a chemical substance secreted from a cell, and the chemical substance secreted by a cell contained in a confinement region of a well Is analyzed by an optical analysis means in a measurement region formed so as to be continuous with the confinement region and prevent cells from entering the inside.

  In the multi-well plate disclosed in the present application, a well forming layer made of a hydrophobic member is laminated on a base plate having a hydrophilic member disposed on at least an upper surface, and the hydrophilic member is formed on the well forming layer. Is a multi-well plate in which a plurality of wells forming a bottom surface are formed, wherein the well is confined to a confinement region formed to be capable of containing a secretory fluid to be measured, and continuous to the confinement region. And a measurement region formed so that the cells do not enter, and the measurement region is arranged in a matrix in the well formation layer.

  The method for analyzing cell secretion disclosed in this application efficiently analyzes non-destructive analytical tests that can be repeatedly analyzed because the cell secretions in the measurement area where the cells of the well cannot enter can be analyzed by optical analysis means. Can do.

  In addition, the multiwell plate disclosed in the present application forms a well having a confinement region and a measurement region in a hydrophobic well formation layer laminated on a hydrophilic base plate with an upper surface. Can be easily accommodated, and contamination of the secretions from the cells between adjacent wells can be prevented, so that accurate and rapid analysis can be performed.

It is an image figure for demonstrating the outline | summary of the analysis method of the cell secretion concerning this embodiment. It is a block diagram explaining the structure of the Raman scattering microscope used for the Raman scattering spectroscopy analysis as an optical analysis means. It is a figure explaining the shape of the multiwell plate concerning this embodiment. FIG. 3A is an enlarged photograph of the actually produced multiwell plate. FIG. 3B is a plan view for explaining the shape of the well. It is an image figure explaining the process of accommodating the cell used as a measuring object in the well of a multiwell plate in the analysis method of the cell secretion concerning this embodiment. FIG. 4A is a diagram showing the appearance of the multiwell plate. FIG. 4B is a diagram illustrating a state in which an aqueous solution containing cells to be measured is supplied onto a multiwell plate. FIG. 4C is a diagram showing a state where the upper surface of the multi-well plate is covered with oil so that the aqueous solution in each well is not mixed. It is a microscope picture which shows the state by which the human breast cancer cell used as a measuring object is confined in the confinement area | region of a well. It is a microscope picture which shows the state which irradiated the laser beam for a Raman scattering spectroscopy analysis to each part in a well in the state by which the cell of the measuring object was accommodated in the confinement area | region. It is a figure which shows the Raman spectrum at the time of performing a Raman scattering spectroscopic analysis in the predetermined position of the measurement area | region of a well, and a comparison area. It is a figure which shows the Raman spectrum obtained from the cell secretion in the predetermined position of the measurement area | region obtained by removing the component of aqueous solution.

  The cell secretion analysis method of the present disclosure is an analysis method for analyzing a chemical substance secreted from a cell, the chemical substance secreted by cells contained in a confinement region of a well being continuous with the confinement region, And in the measurement area | region formed so that the cell cannot enter inside, it analyzes by an optical analysis means.

  By doing so, the region where the secreted product secreted from the cell is present without the presence of the cell is clarified, and the portion to be analyzed can be easily determined by the optical analysis means, so that the analysis throughput is increased. improves. In addition, in order to perform nondestructive inspection using optical analysis means instead of destructive inspection to remove secretions, it is possible to analyze changes over time by repeating analysis at predetermined time intervals, etc. Analysis can be performed. Furthermore, since the analysis is performed by an optical analysis means, necessary data can be acquired without further preparation steps for analysis.

  In the method for analyzing cell secretions according to the present disclosure, the optical analysis means is preferably Raman scattering spectroscopic analysis. By irradiating laser light to a predetermined position in the measurement area by using Raman scattering spectroscopy that can understand the type and composition of molecules from the amount of Raman scattering shift of the laser light irradiated to the object to be measured The state of cell secretion at a specific position can be grasped at a predetermined timing.

  In addition, a comparison region that is not connected to any of the confinement region and the measurement region and that cannot enter the cells is further provided, and the substance in the comparison region is optically analyzed. It is preferable to calibrate the measurement data obtained from the measurement region using the result analyzed by the above. By doing in this way, the data specific to the cell secretion excluding the data depending on the aqueous solution containing the cells can be easily obtained, and more accurate analysis results can be obtained quickly.

  Furthermore, it is preferable that a plurality of wells each having the confinement region and the measurement region are formed in a well plate, and the measurement regions are arranged in a matrix form. By doing so, it is possible to efficiently and statistically analyze the secretions from each of the plurality of cells by the optical analysis means.

  Furthermore, it is preferable that the well is formed of a bottom surface formed by a hydrophilic member and a side surface formed by a hydrophobic member disposed on the bottom surface. By doing in this way, it becomes easy to accommodate the aqueous solution containing the cells in the wells, each well can be easily separated, and contamination of cell secretion can be effectively prevented.

  In this case, it is preferable that the substance in the measurement region is analyzed by an optical analysis means in a state where a hydrophobic liquid is developed above the side surface of the well. By doing in this way, mixing of the aqueous solution accommodated in the adjacent well can be effectively prevented by the hydrophobic liquid developed on the upper surface of the well plate.

  Furthermore, it is preferable to select the measurement region continuous with the confinement region in which one cell is confined, and analyze the cell secretion in the measurement region. By doing in this way, the analysis about the cell secretion from one cell can be performed efficiently.

  Furthermore, the cell secretion in the measurement region can be measured a plurality of times with a predetermined time interval. Moreover, irradiation light can be irradiated to different positions in the measurement region, and the cell secretion can be analyzed using the irradiation light at each position. By doing in this way, more multilateral analysis about a cell secretion can be performed.

  In the multiwell plate of the present disclosure, a well forming layer made of a hydrophobic member is laminated on a base plate having a hydrophilic member disposed on at least an upper surface, and the hydrophilic member has a bottom surface on the well forming layer. A multi-well plate in which a plurality of wells are formed, wherein the well is formed continuously with a confinement region formed so as to accommodate cells secreting a secretion fluid to be measured, and the confinement region. And a measurement region formed so that the cells do not enter, and the measurement region is arranged in a matrix on the well forming layer.

  With this configuration, the multi-well plate according to the present disclosure has a bottom surface of the well made of a hydrophilic member. Therefore, the confinement region contains cells that secrete the secretion fluid to be measured in the confinement region. An aqueous solution containing cells can be easily accommodated in the measurement region. Moreover, it can prevent effectively that the aqueous solution containing a cell mixes between different wells. Furthermore, by arranging the measurement regions in a matrix, it is possible to standardize the movement of the multiwell plate on the analyzer, so that it is possible to achieve a high analysis throughput for a large number of wells.

  In the multi-well plate according to the present disclosure, a comparison region formed so as not to be continuous with the well and to allow the cell to enter is formed in an interval portion of the portion where the measurement region is formed. Is preferred. By doing in this way, since the aqueous solution which contained the cell can be analyzed in the state which is not influenced by the cell and its secretion, the analysis of the secretion from a cell can be performed more correctly.

  Further, the confinement region has a substantially circular shape in plan view, the measurement region has a substantially rectangular shape in plan view formed to extend from the confinement region in one direction, and the comparison region has a direction in which the measurement region extends. It is preferable that it is a substantially rectangular shape in a plan view formed substantially in parallel with each other. In this way, cells that tend to have the same size in the vertical and horizontal directions, such as a circle, can be easily accommodated in the confinement region. In addition, it is possible to prevent cells from entering the measurement area and the comparison area, and it is possible to analyze cell secretions according to the difference in distance from the cells. Furthermore, the confinement region, the measurement region, and the comparison region can be formed on the multi-well plate with high density and with less horizontal movement during analysis.

(Embodiment)
Hereinafter, embodiments of a method for analyzing a cell secretion and a multiwell plate according to the present disclosure will be described with reference to the drawings.

  In the following embodiment, a case where Raman scattering spectroscopic analysis is used as an optical analysis means used in the method for analyzing cell secretions is exemplified.

  FIG. 1 is an image diagram for explaining an outline of a cell secretion analysis method according to the present embodiment.

  FIG. 1 shows a state in which the secretion 3 secreted from the cells 1 in the well 10 formed in the multi-well plate 20 is analyzed using Raman scattering spectroscopy.

  In the cell secretion analysis method according to the present embodiment, the cell 1 is contained in the well 10 in a state of being contained in the culture solution 2 that is an aqueous solution. The well 10 is composed of a confinement region 11 in which the cells 1 are accommodated, and a measurement region 12 formed continuously from the confinement region 11 and into which the cells 1 cannot enter. With the cells 1 being confined in the confinement region 11 as described above, the wavelength characteristics of Raman scattering (scattered by the secretion 3 secreted into the culture solution 2 by irradiating the measurement region 12 with the laser beam 4 ( By examining (Raman shift), the composition and structure of the secretion 3 from the cell 1 can be analyzed.

  In FIG. 2, the example of schematic structure of the Raman scattering microscope used for the analysis method of the cell secretion of this embodiment is shown.

  The Raman scattering microscope 30 used in the cell secretion analysis method according to the present embodiment is a so-called spontaneous Raman scattering microscope, and the laser light 4 generated by the laser light source 31 is magnified by the magnifying lens system 32, The light is reflected by the total reflection mirror 33 and the dichroic mirror 34 and can be deflected two-dimensionally by a galvanometer mirror 35 which is a deflecting device. Further, the laser beam 4 is introduced into the microscope case by the relay lens system 36, guided on the optical axis of the microscope by the prism mirror 37, and irradiated on the sample 39 through the objective lens 38.

  In this embodiment, the laser beam 4 is irradiated so as to be focused in the measurement region 12 of the well 10 formed on the multiwell plate 20 shown in FIG. Note that the galvanometer mirror 35 serving as a beam deflecting device and the rear focal plane of the sample 39 are set so as to be optically conjugate, and the laser beam 4 is directed in a direction perpendicular to the optical axis thereof, that is, The laser beam 4 can be focused on a predetermined plane position in the measurement region 12 of the well 10 while observing the field of view of the microscope while scanning in the direction of the main surface of the multi-well plate 20 at a high speed. The point can be moved.

  The Raman scattered light 5 generated from the secretion 3 from the cell 1 existing at the focus position of the laser light 4 is collimated by the objective lens 38 and irradiated to the dichroic mirror 34 through the relay lens system 36 and the galvanometer mirror 35. Is done. The Raman scattered light 5 whose optical path is branched by the dichroic mirror 34 travels straight as it is, passes through the excitation light removal filter 40 and the total reflection mirror 41, is focused by the focusing lens 42, and is introduced into the spectroscope 43. The Raman scattered light 4 dispersed by a spectroscope 43 having a dispersive spectroscopic optical system such as a diffraction grating or a prism, or a Fourier transform type spectroscopic optical system is converted into one-dimensional or two-dimensional by a highly sensitive CCD camera 44. Detect as image data.

  The relationship between the Raman shift frequency and the Raman scattering intensity can be obtained from the spectral characteristics of the Raman scattered light generated by the cell secretion that is the object to be measured, and the composition of the cell secretion is obtained. Can be analyzed.

  In the Raman scattering microscope 30 shown in FIG. 2, the laser light source 31 for irradiating the laser beam 4 is a solid-state laser in a wavelength region (wavelength 200 nm to 1100 nm) extending from the deep ultraviolet region to the visible light region and the near infrared region. Can be used. Note that the spectrum width is narrow, the brightness is high, and a CW laser (Continuous wave laser) is preferable. As an example, a YAG laser having a wavelength of 532 nm can be suitably used as a laser light source. For example, when a Raman scattering microscope is used for imaging of living cells, etc., there is a possibility that the observation target cell may be damaged if laser light having a short wavelength is used. However, the method for analyzing a cell secretion shown in the present embodiment In this case, since cells do not enter the measurement region irradiated with the laser light, it is possible to use a laser light source that irradiates light in the deep ultraviolet region with a short wavelength.

  In addition, as shown in FIG. 2, the objective lens 38 of the microscope portion is a portion that irradiates the laser light 4 and receives the Raman scattered light 5, so that in order to obtain high light collection efficiency and detection efficiency, It is preferable to use a high numerical aperture lens having a numerical aperture (NA) of 0.7 or more.

  Note that the specific configuration of the Raman scattering microscope 30 shown in FIG. 2 used in the cell secretion analysis method according to the present embodiment is merely an example. For example, a general Raman scattering microscope including a commercially available Raman scattering microscope such as using a beam splitter instead of the dichroic mirror 34 shown in FIG. 2 as a means for branching the laser light 4 and the Raman scattered light 5. Can be used. In particular, the arrangement of various lens members and members for changing an optical path such as a mirror and a prism can be configured in various forms.

  In the cell secretion analysis method according to the present embodiment, the well 10 formed in the multi-well plate 20 includes a confinement region 11 in which the cells 1 are accommodated and a measurement region 12 in which the cells 1 cannot enter. It is configured.

  FIG. 3 shows the configuration of a multiwell plate used in the method for analyzing cell secretions according to this embodiment. FIG. 3A shows an enlarged photograph of the multiwell plate actually created by the inventors. FIG. 3B is a plan view for explaining the shape of the well formed in the multi-well plate.

  As shown in FIG. 3A, the confinement region 11 of the well 10 formed in the multi-well plate 20 is formed in a substantially circular shape when viewed in plan, and the measurement region 12 is formed from a part of the confinement region 11. It is formed in a substantially rectangular shape extending in one direction.

  By forming the well 10 in such a shape that the confinement region 11 and the measurement region 12 are combined, it can be generally grasped as a substantially spherical shape or a substantially disc shape, that is, when viewed in a plan view, The cell 1 having a relatively small difference between the size and the lateral size can be effectively prevented from entering the measurement region 12, and the Raman scattering measurement is performed in the measurement region 12 as shown in FIG. By irradiating the laser beam 4 for performing the above, the secretion 3 secreted from the cell 1 can be analyzed in a state clearly distinguished from the cell 1.

  The specific size of the well 10 is determined according to the size of the cell to be measured. For example, the well 10 is obtained from a substantially spherical human breast cancer cell (MDA-MB-231) having a diameter of about 25 μm. When analyzing secretions, the diameter of the circular portion of the confinement region 11, that is, the values of A and B in FIG. 3B are both 40 μm, and the length portion of the measurement region 12 in FIG. The value of C was 40 μm, and the value of D in FIG. 3B which is the width of the measurement region 12 was 20 μm. In this way, each cell can be accommodated in the confinement region 11, and a situation where the cell 1 cannot enter the measurement region 12 can be created.

  In addition, the measurement area | region 12 can prevent the penetration | invasion of the cell 1 to an inside by making the value of the width | variety D into the diameter size of the cell 1 or less. In this respect, the degree of freedom in designing the length C of the measurement region 12 is high, but in designing the well 10 shown in the present embodiment, the degree of diffusion of the secretion 3 from the cells 1 accommodated in the confinement region 11. Is designed so that the length C of the measurement region 12 is larger than the width D.

  In the cell secretion analysis method according to the present embodiment, the secretion in the measurement region is analyzed by Raman scattering spectroscopy as an optical analysis means. By using optical analysis means such as Raman scattering spectroscopic analysis, it is possible to analyze secretory components and the like without affecting the environment in the well including the measurement region.

  By utilizing this, for example, it is possible to repeatedly analyze secretions at a predetermined time interval from the start of analysis, and to observe changes in the composition of secretions secreted from cells over time. Can do. In addition, as described above, in the cell secretion analysis method according to the present embodiment, the Raman scattering microscope that performs the Raman scattering spectroscopic analysis includes the galvanometer mirror as the beam deflecting unit. A desired position can be irradiated by being deflected in the measurement region. In this way, by changing the irradiation position of the laser beam for performing Raman scattering spectroscopic analysis within the measurement region, the composition of the secretion can be changed according to the change in the distance from the confinement region where the cells are confined. It is possible to observe.

  As shown in FIG. 3A, in the multiwell 20 used in the cell secretion analysis method according to the present embodiment, the measurement regions 12 of the wells 10 are parallel to each other, and both end positions thereof are in a straight line. They are arranged in a line. The measurement regions 12 of each well 10 are arranged in a matrix so as to be arranged at a predetermined interval in a direction orthogonal to the arrangement direction.

  As described above, the measurement regions 12 are arranged in parallel with each other and arranged in a row in which the lateral positions are aligned, and are also arranged in the row direction at predetermined intervals, thereby sequentially measuring the measurement regions of different wells 10. In this case, the direction in which the multiwell plate 20 is moved with respect to the optical axis of the Raman scattering microscope can be limited to two directions, the vertical direction and the horizontal direction. A microscope such as a Raman scattering microscope generally includes an xy stage that can move a plate on which a sample is placed in the x direction and the y direction. For this reason, by arranging the measurement regions 12 to be measured on the multi-well plate 20 in a matrix as described above, a plurality of measurements are arranged using the xy stage provided in the Raman scattering microscope. It becomes easy to automatically control and move the region sequentially to the optical axis position of the Raman scattering microscope, and the analysis throughput for a large number of samples can be improved.

  As shown in FIG. 3 (a), in the multiwell plate 20 used in the method for analyzing cell secretions according to the present embodiment, the measurement region 12 and the measurement region 12 are substantially positioned at an intermediate position between the measurement regions 12. The same substantially rectangular comparison region 24 is arranged so that its length direction is parallel to the length direction of the measurement region 12.

  The comparison region 24 has a width that is the same as the width D of the measurement region 12 (in the case of FIG. 3A, 20 μm). Can be formed as a region that cannot enter. The comparison region 24 is provided, and the culture solution 2 containing the cells 1 at the same time as the well 10 is accommodated in the comparison region 24, so that the influence of the secretion 3 from the cells 1 in the comparison region 24. It can be set as the state in which the culture solution 2 of the state which has not received is accommodated.

  For this reason, the analysis data of the culture solution 2 in the measurement region 12 can be directly obtained by performing the Raman scattering spectroscopic analysis on the secretion solution 2 in the comparison region 24 using the Raman scattering microscope in the same manner as the measurement region 12. Can do. The obtained analysis data of the culture fluid 2 alone is arithmetically subtracted from the analysis data in the measurement region 12 to obtain the analysis data of the cell secretion 3 in a state in which the influence of the components of the culture solution 2 is excluded. Can be obtained.

  FIG. 3A shows an example in which the comparison regions 24 are arranged in the interval portions of all the measurement regions 12 as the multiwell plate 20 of the present embodiment. By doing in this way, the analysis data of the culture solution 2 in the comparison area | region 24 can be obtained suitably, without moving a multiwell plate largely as needed. However, as described above, in the comparison region 24, the main purpose is to obtain analysis data that serves as a reference for judging the state of the culture solution in the measurement region 12, and therefore, the same number as the measurement region 12 is necessarily required. It is not a thing. For example, if no change has occurred in the components of the culture solution 2 in all the regions of the multiwell plate 20, it is sufficient if one comparison region 24 is formed on the multiwell plate 20. Even when the condition of the culture solution 2 is assumed to be different depending on the portion of the multiwell plate 20 because the external environment such as the incident state of light rays and the like on the multiwell plate 20 and the temperature distribution are different. By arranging one comparison region 24 for each of, for example, 10 to several tens of measurement regions 12, it is possible to achieve the original significance that the comparison region 24 should fulfill.

  In the multi-well plate 20 used in the cell secretion analysis method according to the present embodiment, the bottom surface 13 of the well 10 is composed of a hydrophilic member, and the side surface 14 of the well 10 is composed of a hydrophobic member.

  When the culture solution 2 containing the cells 1 is introduced into the well 10, it is preferable that the bottom surface 13 of the well 10 is hydrophilic. On the other hand, after the cells 1 and the culture medium 2 are accommodated in the wells 10, the culture medium 2 is mixed between the adjacent wells 10, and the secretion 3 from one cell 1 cannot be analyzed accurately. It is necessary to prevent it. For this reason, in the cell secretion analysis method of this embodiment, as shown in FIG. 1, the upper surface of the well 10 and the upper surface of the multiwell plate 20 located around the well 10 are covered with hydrophobic oil 23. I am doing so. At this time, the side surface 14 of the well 10, in particular, the vicinity of the upper end portion of the side surface 14 and the upper surface portion around the well 10 are made of a hydrophobic member, so that the culture solution 2 is eliminated and the well 10 is closely attached In this state, the oil 23 can be supplied.

  Here, the culture solution 2 containing the cells 1 is supplied onto the multi-well plate 20 to supply the culture solution 2 containing the cells 1 in the confinement region of the well 10, and the measurement region of the well 10 and the comparison region described above. The step of supplying the culture solution 2 into the medium 24 and the step of covering the upper surface of the formation surface of the well 10 with the oil 23 will be described.

  FIG. 4 is an image diagram for explaining a culture solution containing cells in a multiwell plate and a step of supplying oil covering the upper surface thereafter.

  FIG. 4A shows a state in which the multiwell plate 20 is viewed obliquely from above in order to explain the configuration of the multiwell plate 20 according to the present embodiment.

  As shown in the cross-sectional configuration of FIG. 1, in the multi-well plate 20 according to the present embodiment, a well forming layer 22 made of a hydrophobic resin material such as a fluororesin is laminated on a base plate 21 made of hydrophilic glass. Configured. Each well 10 and the comparison region 24 can be easily formed in the well forming layer 22 by a photolithography method using a pattern mask depicting a desired opening shape with respect to the well forming layer 22.

  By accurately etching the thickness of the well formation layer 22 by photolithography, the upper surface 21a of the base plate 21 is exposed, and the bottom surface 13 of the well 10 formed of a hydrophilic member can be configured. Further, since the well forming layer 22 is formed of a hydrophobic resin member, the side surface 22a in which the pattern of the well 10 is etched and the upper surface 22b other than the formation portion of the well 10 are hydrophobic, and the hydrophobic A side surface 14 of the well 10 formed of a member can be formed.

  In the above, an example in which the base plate 21 constituting the multi-well plate 20 is made of glass that is a hydrophilic member has been shown. However, the base plate 21 has at least an upper surface 21a that is hydrophilic. For example, only the upper surface of the hydrophobic glass plate may be processed to have hydrophilicity.

  As shown in FIGS. 3A and 3B, in the multiwell plate 20 according to the present embodiment, the diameter of the circular confinement region 11 is 40 μm, the width of the measurement region 12 is 20 μm, and the length is long. The thickness was 40 μm. The interval between the adjacent wells 10 is 40 μm between the confinement regions 11, that is, the arrangement pitch of the wells 10 is 80 μm. Furthermore, a comparison region 24 (length: 60 μm) having a width of 20 μm was arranged in parallel with the measurement region 12 in the space between adjacent wells 10 to form a row of wells 10. The interval between the column of the wells 10 and the column of the well 10 located next to each other is such that the interval between the confinement region 11 and the measurement region 12 in the adjacent column is 30 μm, that is, the arrangement pitch of the wells 10 in the row direction is The thickness is set to 110 μm. The size and shape of the wells 10 on the multi-well plate 20 and the arrangement pitch thereof should be appropriately designed as appropriate based on the size and shape of the cells secreting the secretion fluid to be measured as described above. It is.

  Although not shown, the shape and size of the multiwell plate 20 itself is a circle having a diameter of about 0.5 cm to 2.5 cm or a square having a side of 0.5 cm to 2.5 cm. Can do. The outer shape and shape of the multi-well plate 20 are determined within a range that can be used for optical means for analyzing the secretion 3 in the measurement region 12. In the case of the present embodiment, the size is appropriately determined in consideration of the number of wells 10 to be formed and the arrangement interval among the sizes that can be placed on the xy stage used in the Raman scattering microscope 30.

  Returning to FIG. 4, as shown in FIG. 4B, the suspension 2 containing the cells 1 is supplied to the upper surface of the multiwell plate 20. Although illustration in FIG. 4 is omitted, a peripheral wall portion formed upward from the surface of the multiwell plate 20 is formed in the peripheral portion which is the outer shape portion of the multiwell plate 20. For this reason, as shown in FIG.4 (b), the suspension 2 can be supplied so that the upper surface of the multiwell plate 20 may be covered with the suspension 2 of predetermined thickness. Then, in the suspension 2, the cells 1 are left for a while until they settle on the surface of the well forming layer 22 or in the confinement region 11 of the well 10.

  At this time, as shown in FIG. 4B, a part of the cell 1 enters the confinement region 11 of the well 10. On the other hand, the remaining cells 1 remain on the upper surface of the well forming layer 22. The number density of the cells 1 in the culture solution 2 is adjusted so that the cells 1 enter the confinement region 11 at a certain ratio or more.

  The culture solution 2 is contained in the well 10 and the comparison region 24, and the culture solution 2 remaining on the upper surface of the well forming layer 22 in a state where the cells 1 are accommodated in the confined region 11 in a predetermined ratio, and the culture solution 2 is contained therein. In order to remove the cells 1, an oil 23 as a hydrophobic liquid is supplied to the upper surface of the well forming layer 22.

  As described above, since the well forming layer 22 is formed of a hydrophobic member, the oil 23 is slowly supplied from the vicinity of the end when the well forming layer 22 is viewed in plan view. The oil 23 having high wettability spreads so as to push away the culture solution 2 and covers the upper surface 22b (see FIG. 1) of the well forming layer 22.

  The culture solution 2 contained in the well 10 and the comparison region 24 remains in the well 10 and the comparison region 24 because both the bottom surface 11 of the well 10 and the bottom surface of the comparison region 24 are formed of hydrophilic members. be able to. In this way, as shown in FIG. 4C, the respective wells 10 and the comparison region 24 are both arranged independently of the other portions, that is, the culture solution 2 between each other. In the well 10 in which the cells 1 are contained, the secretion 3 from the cells 1 can be accurately analyzed in the measurement region 12.

  FIG. 5 shows a photomicrograph of a well in a state where cells are accommodated.

  It can be confirmed that the cell 1 is accommodated in the confinement region 11 of the well 10 and the confinement region 11 and the measurement region 12 are filled with the culture solution 2.

  In this state, the measurement region 12 is irradiated with the laser beam 4 of the Raman scattering microscope 30 to analyze the secretion in the measurement region 12.

  As described above, in the cell secretion analysis method according to the present embodiment, the culture solution 2 containing the cells 1 is supplied onto the multiwell plate 20 so that the cells 1 enter the confinement region 11 of the well 10. It is trying to settle naturally. For this reason, for example, it is conceivable that the cell 1 is not contained in the confinement region 11 of about half. The confinement region 11 is designed to have a shape and size so that one cell 1 can be accommodated, but in rare cases, two (very rarely more) cells enter the confinement region. In some cases.

  Therefore, in the cell secretion analysis method according to the present embodiment, the Raman scattering measurement is not performed on the measurement region 12 of the well 10 in which such one cell is not accommodated in the confinement region 11. The measurement throughput for one multi-well plate 20 is improved.

  Whether the cells are not contained in the confinement region of the well or the state where two cells are contained is enlarged by a low magnification microscope as necessary, It can be judged by visual recognition. In addition, using a predetermined program, image recognition processing is performed on the image of the surface of the multiwell plate at a constant magnification, the measurement area to be measured is narrowed down, and automatically linked with the control of the xy stage. In other words, it is possible to collect data by Raman scattering measurement only in a necessary measurement range.

  Thus, after grasping | ascertaining the measurement area | region used as a measuring object, a Raman scattering spectroscopy analysis is performed with respect to the accommodated secretion.

  Using the Raman scattering microscope shown in FIG. 2, the laser light 4 is condensed at a predetermined position in the measurement region 12, and the Raman scattered light 5 generated at this time is dispersed by the spectroscope 43, and the image data is obtained by the camera 44. Record and perform spectral analysis.

  When the irradiation position (focal position) of the laser beam 4 is close to the side wall portion of the measurement region 12, Raman scattering of the organic molecules of the resin constituting the well forming layer 22 is excited and becomes measurement data noise. For this reason, by adjusting the galvanometer mirror 35, the laser beam 4 is condensed at a position away from the side wall portion of the measurement region 12 as an example by 3 μm or more, more preferably about 5 μm.

  Similarly, in order to avoid measuring Raman scattering due to the component of the glass constituting the bottom surface 13 of the measurement region 12 and the component of the oil 23 covering the top surface of the measurement region 12, the thickness ( It is considered that the focal position of the laser beam 4 in the (depth) direction is preferably 3 μm or more, more preferably about 5 μm away from the upper and lower boundaries of the measurement region.

  FIG. 6 is a photomicrograph taken in a Raman scattering microscope used in the method for analyzing cell secretions according to the present embodiment, in a state where a predetermined position in the well is irradiated with laser light.

  A model showing the irradiation position of the laser beam 4 is shown in the upper left of FIG. In the model, the numbers written in the well 10 indicate the irradiation position of the laser beam 4 in the photograph arranged on the right side of FIG. Since the Raman scattering microscope 30 used in the cell secretion analysis method of the present embodiment includes the deflecting device using the galvanometer mirror 35 as described above, the laser beam 4 is accurately irradiated to a desired position in the well 10. can do.

  In addition, it can be seen from the photograph shown as “3” in FIG. 6 that the cell 1 is located substantially in the center of the confinement region 11. This state is indicated by a dotted line in the upper left drawing of FIG. Since the cell 1 is present in the substantially central portion of the circular confinement region, the laser beam 4 is irradiated to the cell 1 in the well 10 in the photograph where the measurement locations are shown as “1” and “2” in FIG. It is thought that. Therefore, in analyzing the secretion from the cell 1, the irradiation positions indicated by “1” and “2” are not preferable.

  On the other hand, when analyzing the cells themselves together with the secretions from the cells, it is only necessary to irradiate the confinement region with laser light as shown in the photographs shown as “1” and “2” in FIG. Thus, according to the method for analyzing a cell secretion according to the present embodiment, by appropriately adjusting the irradiation position of the laser light in the well in which the cell is accommodated, not only the secretion from the cell, It can be seen that the cell itself can be analyzed at the same time.

  In addition, the analysis method of the cell secretion concerning this embodiment calculates | requires the component by irradiating a laser beam to the measurement area | region in which the secretion from a cell is accommodated, and using a Raman scattering spectroscopy analysis. The analysis by Raman scattering spectroscopy is performed by analyzing the data of the Raman scattered light generated by irradiating the laser beam. For example, unlike the mass analysis used in the analysis method described as the prior art, the analysis There is no need to perform any special pre-processing for the purpose. For this reason, in the Raman scattering microscope, the measurement area in the multi-well plate is placed at the position where the irradiation light is irradiated in the Raman scattering microscope using an xy stage or the like, and the cell secretion in each measurement area is immediately Can be analyzed. As a result, according to the cell secretion analysis method according to the present embodiment, data analysis of desired measurement regions can be performed one after another on a multiwell plate in which a large number of measurement regions are formed. Especially when it is necessary to perform typical data analysis, it is possible to perform measurement with high throughput.

  In addition, the optical analysis method is a non-destructive analysis method that is not a so-called destructive inspection so that the culture solution is collected and performed as in the prior art, and the same measurement object is repeatedly measured. It can be carried out.

  For this reason, after one cell is confined in the well, the change over time of the secretion secreted from the cell can be measured by repeating the measurement at regular intervals.

  In addition, the position of laser light irradiation can be moved appropriately for analysis within the measurement area, and measurements can be performed repeatedly, so the diffusion speed and secretion diffusion direction can be grasped. For example, it is possible to perform a multi-dimensional measurement with a two-dimensional spread, such as creating a diffusion map of secretions according to the distance from the cell.

  FIG. 7 and FIG. 8 show spectra of measurement results actually performed using a Raman scattering microscope 30 for a secretion from one cell. The human breast cancer cells (MDA-MB-231) described above were used as the measurement target cells.

  FIG. 7 shows spectra obtained by irradiating three places in the measurement region and one place in the comparison region with laser light.

  Moreover, FIG. 8 has shown the data which subtracted the Raman scattering spectrum performed with respect to each of the Raman scattering spectrum in three places of a measurement area | region with respect to the culture solution in a comparison area | region.

  In FIG. 7, a spectrum indicated by a thick solid line as reference numeral 51 is a Raman scattering spectrum for the culture solution in the comparison region 24. Reference numeral 52 denotes the Raman scattering spectrum at the position “3” in the laser light irradiation position model diagram in FIG. 6, and reference numeral 53 denotes the laser light 4 at the position “4” in the irradiation position model diagram in FIG. Reference numeral 54 denotes a Raman scattering spectrum when irradiated with the laser beam 4 at a position “5” in the irradiation position model diagram of FIG. 6.

As shown in FIG. 7, the characteristics of the spectrum indicated by reference numerals 52, 53, and 54, which are data in the measurement region, and the characteristics of the spectrum indicated by reference numeral 51, which is the measurement data in the comparison region 24, are clearly different in tendency. I can understand that. Further, in the spectrum data shown in FIG. 7, in any measurement data, there is a relatively strong output peak in the portion where the wavelength (cm ⁻¹ ) is 1200 to 1400. This peak is considered to be due to the influence of the Raman scattering spectrum based on the component of the oil 23 covering the upper surface of the multiwell plate 20.

  In contrast, FIG. 8 shows the Raman scattering spectrum obtained by subtracting the analysis data of the comparison region 24 from the Raman scattering spectrum at each irradiation position of the laser beam 4. 8, reference numeral 55 indicates a Raman scattering spectrum at the position “3” in the laser light irradiation position model diagram in FIG. 6, that is, a spectrum obtained by subtracting the data of reference numeral 51 from the data of reference numeral 52 of FIG. . Similarly, the reference numeral 56 in FIG. 8 indicates the Raman scattering spectrum when the laser beam 4 is irradiated at the position “4” in the irradiation position model diagram in FIG. 6 (the reference numeral 53 in FIG. 7 minus the reference numeral 51). Reference numeral 57 denotes a Raman scattering spectrum (the reference numeral 54 in FIG. 7 minus the reference numeral 51) when the laser beam 4 is irradiated at the position “5” in the irradiation position model diagram of FIG.

  From the spectrum data shown in FIG. 8, an output peak that appears to indicate a benzene ring near a wavelength of 1000, an output peak that indicates a C═C bond near a wavelength of 1600, and a C═O bond near a wavelength of 1750 are shown. It can be seen that an output peak appears.

  It should be noted that an error such as a negative data has occurred in the wavelength portion considered to indicate the oil appearing in FIG. In FIG. 7, the influence of oil is strong, and the negative data appears in FIG. 8. This is because the depth of the well used for the measurement of the current data shown in FIGS. It is considered that the focal position of the irradiation light from the Raman scattering microscope was not sufficiently separated from the oil located on the upper surface of the well because it was relatively shallow. If the secretion from the cells that should be measured originally contains a component that has a peak at the position where the oil-derived spectrum occurs, set the focal position of the laser beam at a position sufficiently away from the oil. Allows more accurate analysis. If necessary, use oils with different compositions so that oil-derived peaks do not occur in the region where the wavelength peaks of the components to be measured appear even if oil-derived peaks occur. be able to.

  In addition, by using a multi-well plate in which wells are arranged in a matrix in the vertical and horizontal directions as shown in FIG. 3A, a secreted product secreted from each cell for a plurality of cells. Statistical analysis can be performed.

  In this case, as an example, first, the first well is placed at the center of the field of view of the Raman scattering microscope using the xy stage. At this time, it is effective to specify in advance the detailed position in each well, such as the center of the well or the boundary between the confinement region and the measurement region, using the two-dimensional xy coordinates.

  If necessary, the position of the multiwell plate can be finely adjusted while confirming the field of view of the actual microscope. This fine adjustment can be automatically performed using the image recognition means.

  Next, the confinement region of the well is observed to confirm whether one cell is accommodated therein. If no cells are contained, or if two (or more) cells are contained, the well is not analyzed and the position of the multiwell plate is changed for the next well analysis. To do. When one cell is accommodated, a predetermined position in the measurement region is irradiated with laser light, and the obtained Raman scattering spectrum data is measured and recorded.

  When analyzing the state of the secretion at another position in the measurement area at the same timing, the beam irradiation position is moved by the deflecting means, the beam is irradiated again, and the spectrum data of the resulting Raman scattered light is measured. ,Record.

  At this time, the xy stage is operated to irradiate the adjacent comparison region with laser light to acquire culture solution analysis data. In many cases, it is not necessary to perform the measurement of the comparison data every time the measurement is performed in the measurement region, and the comparison data can be appropriately obtained at a timing when a plurality of measurements or a predetermined time elapses.

  By performing such measurement on a plurality of wells formed by computer control, for example, it is possible to perform statistical analysis of secretions for a large number of cells.

  When measuring secretions over time, the above measurement may be repeated every time a predetermined time elapses.

  The obtained Raman scattering spectrum can be used for data analysis after removing noise using the comparison data. For example, the composition of cell secretion can be analyzed based on the peak position of Raman scattered light and its intensity. In this case, more accurate secretion analysis can be performed by performing statistical analysis based on a large number of data.

  In addition, when a secretion is measured over time or when a two-dimensional analysis such as a two-dimensional spread of the secretion is performed, a predetermined substance is assigned a specific color based on the obtained data. For example, the analysis result can be expressed as an image that can be easily understood.

  As described above, the cell secretion analysis method disclosed in the present application and the embodiment of the multi-well plate suitably used for this analysis method have been described with reference to the drawings.

  According to the cell secretion analysis method shown in this embodiment, the composition of a secretion secreted from one cell is analyzed by Raman scattering spectroscopy as an optical analysis means that is a non-destructive analysis method. be able to. At this time, by separating the confinement region where the cells are confined and the measurement region where data analysis is performed, highly accurate data can be obtained with high throughput. Further, since an optical analysis means that is a non-destructive inspection is used, it is possible to repeatedly analyze one sample.

  The multi-well plate disclosed in the present application is configured by distinguishing a confinement region for confining cells and a measurement region, and further, the bottom surface of the well is formed of a hydrophilic member and the side surface of the well is formed of a hydrophobic member. The step of confining the cells contained in the culture solution in the confinement region and the step of measuring the secretion from the cells confined in the confinement region can be easily and reliably performed.

  In the above-described embodiment, a method of performing spectroscopic analysis using spontaneous Raman scattering has been described as an optical analysis means for cell secretion. However, the optical analysis method used in the cell secretion analysis method disclosed in the present application is not limited to a method using spontaneous Raman scattering. Even when using Raman scattering, cell secretion is analyzed by CARS spectroscopic analysis using a coherent anti-Stokes Raman scattering (CARS) microscope, depending on the object to be measured. May be more advantageous. It is also assumed that analysis can be performed using a stimulated Raman scattering (SRS) microscope.

  Furthermore, it is not limited to Raman scattering spectroscopic analysis. For example, various optical analysis methods that are non-destructive component analysis methods such as fluorescence observation of proteins using fluorescent markers and component analysis methods using antibody staining are used. Thus, cell secretions in the measurement region can be analyzed.

  In the above-described embodiment, an example in which cells are confined and a measurement region is secured using a well in which a circular confinement region and a substantially rectangular measurement region are continuously formed has been described. However, the shapes of the confinement region and the measurement region are not limited to those exemplified above, and it is needless to say that the confinement region and / or the measurement region can be configured in a shape different from the above example. In addition, a barrier between the confinement region and the measurement region that is different in width and depth from other parts is configured so that cells do not enter the measurement region, and the depth of the measurement region is confined. By forming it shallower than the region, it is possible to prevent the cells from entering the measurement region. As described above, the confinement region in which cells can be accommodated and the measurement region in which cells cannot enter can be configured in various forms.

  In addition, a circular confinement region is provided at both ends, and a well having a shape similar to a dumbbell in plan view, such as two confinement regions connected by a thin rectangular measurement region, is the same in two confinement regions. Alternatively, it is conceivable to confine different types of cells and measure the composition of the secretion, the spread state, and the change over time due to the interaction between the two cells. Thus, by analyzing the shape of the well formed in the multi-well plate, it is possible to analyze a variety of cell secretions.

  In the above embodiment, the example of analyzing the secretion from one cell confined in the confinement region has been described. However, the shape and size of the confinement region are devised and two or more predetermined numbers of cells are analyzed. Alternatively, by allowing the cell mass to be accommodated in the confinement region, it is also possible to analyze a plurality of cells and secretions from the cell mass.

  Further, in the above embodiment, the upper surface of the multi-well plate is a hydrophobic liquid in order to prevent mixing of the culture liquid between adjacent wells after accommodating the culture liquid containing cells in each well. Explained covering with oil. As described above, by devising the types of members that make up the multi-well plate, the problem of mixing the culture solution in adjacent wells can be solved with a simple operation. In the case of analysis, it is also known that the oil components may adversely affect the analysis results.

  Therefore, as a method of reliably removing the culture solution on the upper surface of the multiwell plate, for example, a method of removing the culture solution on the multiwell plate by wind pressure, or using a blade whose surface is coated with a hydrophobic member Use other methods, such as scraping off the culture solution on the multi-well plate, so that the culture solution does not remain on the surface of the multi-well plate. There is a possibility that analysis can be performed.

  In addition, as an aqueous solution accommodated in a well with a cell, the culture solution suitable for each cell can be used. It is also possible to use a buffer solution. However, when analyzing secretions by Raman scattering spectroscopy, it is not preferable to use a culture medium having a coloring property such as phenol red because it emits light stronger than Raman scattered light in the visible light region. .

  In addition, when analyzing secretions in the measurement region by Raman scattering spectroscopy, surface plasmons are generated by embedding metal nanoparticles in the bottom of the well, and so-called surface enhancement is performed, which performs highly sensitive Raman scattering spectroscopy analysis. It is also possible to use a technique called Raman scattering (SERS).

  The cell secretion analysis method disclosed in the present application is a non-destructive method for the secretion from cells, and realizes high-throughput and high-precision analysis. In addition, the multiwell plate disclosed in the present application is useful for analysis of cell secretions. For this reason, the cell secretion analysis method disclosed in the present application and the multiwell plate can be expected to contribute to research in various fields including the life science field.

1 cell 2 culture solution (aqueous solution)
3 secretions (chemical substances)
4 Laser light 5 Raman scattered light 10 Well 11 Confinement region 12 Measurement region 20 Multiwell plate 21 Base plate 22 Well formation layer

Claims (12)

An analysis method for analyzing chemical substances secreted from cells,
The chemical substance secreted by the cells contained in the confinement region of the well is analyzed by optical analysis means in a measurement region formed so as to be continuous with the confinement region and so that the cells cannot enter the inside. Analysis method of cell secretion.   The method for analyzing a cell secretion according to claim 1, wherein the optical analysis means is Raman scattering spectroscopy. A comparison region formed so that the cells cannot enter the confinement region and the measurement region, and is not connected to the measurement region;
The method for analyzing a cell secretion according to claim 1 or 2, wherein the measurement data obtained from the measurement region is calibrated using a result obtained by analyzing the substance in the comparison region by an optical analysis means.   The cell secretion solution according to any one of claims 1 to 3, wherein a plurality of the wells each having the confinement region and the measurement region are formed in a well plate, and the measurement regions are arranged in a matrix form. Analysis method.   The cell according to any one of claims 1 to 4, wherein the well is formed of a bottom surface formed of a hydrophilic member and a side surface formed of a hydrophobic member disposed on the bottom surface. Analysis method of secretions.   6. The method for analyzing a cell secretion according to claim 5, wherein a substance in the measurement region is analyzed by an optical analysis means in a state where a hydrophobic liquid is developed above the side surface of the well.   The cell secretion solution according to any one of claims 1 to 6, wherein the measurement region continuous with the confinement region in which one cell is confined is selected and the cell secretion solution in the measurement region is analyzed. Analysis method.   The method for analyzing a cell secretion solution according to any one of claims 1 to 7, wherein the cell secretion solution in the measurement region is measured a plurality of times at predetermined time intervals.   The method for analyzing a cell secretion according to any one of claims 2 to 8, wherein irradiation light is irradiated to different positions in the measurement region, and the cell secretion is analyzed using the irradiation light at each position. . A well forming layer made of a hydrophobic member is laminated on a base plate having a hydrophilic member disposed on at least the upper surface, and a plurality of wells in which the hydrophilic member forms a bottom surface are formed on the well forming layer. Multi-well plate,
A confinement region in which the well is formed so as to accommodate a cell that secretes a secretory fluid to be measured; a measurement region formed continuously in the confinement region and so as not to enter the cell; With
The multi-well plate, wherein the measurement regions are arranged in a matrix on the well formation layer.   11. The multi-well plate according to claim 10, wherein a comparison region formed so as not to be continuous with the well and into which the cells cannot enter is formed in an interval portion between portions where the measurement region is formed. .   The confinement region has a substantially circular shape in plan view, the measurement region has a substantially rectangular shape in plan view formed by extending from the confinement region in one direction, and the comparison region is substantially in the extending direction of the measurement region. The multiwell plate according to claim 11, which is formed in parallel and has a substantially rectangular shape in plan view.